Scientists have described stoneflies as one of the most threatened insect groups, one that has experienced high extinction rates in recent decades.

The rediscovery of a fly species in Wales hints at environmental DNA sampling’s potential to change endangered species protection. (Adam McCauley/The New York Times)

Just a few years ago, it seemed like the scarce yellow sally stonefly had gone locally extinct.

In 1995, ecologists collected a single specimen of the aquatic insect in the River Dee near the Wales-England boundary, the species’ only known refuge. For the next two decades, every survey there failed to find another of the stonefly, which is only about a half an inch long.

“There had been so much work done to refind this beast,” said Craig Macadam, conservation director at the Invertebrate Conservation Trust, more commonly known as Buglife, a charity in Britain. “We were all beginning to give up hope.”

Small, isolated populations of stoneflies reside in pristine brooks, where they are vulnerable to pollution and habitat fragmentation. Scientists have described stoneflies as one of the most threatened insect groups, one that has experienced high extinction rates in recent decades.

Even among the numerous species of its family, the scarce yellow sally stonefly (“scarce” is part of its name) is noted for its rarity, said John Davy-Bowker, a freshwater biologist who has surveyed the insect’s population since the 1990s. Without any new evidence of its survival in the River Dee, the scarce yellow sally stonefly would be declared locally extinct, Macadam said; it already had vanished from an assortment of European countries.

“When you actually see the animal alive in front of you and then the next year it’s gone, you feel like you’ve watched it disappear from Earth,” Davy-Bowker said. “Nobody could find it, so that was it. It just disappeared.”

But Davy-Bowker wouldn’t quit. In March 2017, during the season when the River Dee is at its coldest and deepest and stonefly nymphs are large, he put on chest waders and went in.

The results of his search, and how they were then combined with a powerful technology called environmental DNA sequencing, created new hope for an insect that appeared to be gone forever. The rediscovery of this stonefly also suggests how the technique might contribute to efforts to save some of the world’s most critically endangered organisms.

Genetic sleuthing

Environmental DNA, or eDNA, has changed the way conservationists study the environment, improving their ability to keep tabs on species too elusive or imperilled to monitor with traditional methods.

“We know from ‘CSI’ that we leave DNA everywhere,” said Sean Rogers, a biologist at the University of Calgary who published a review of eDNA technology in November. “With conservation, it became — instead of taking nets to try to capture fish or hoping to catch something on a wildlife camera — let’s take forensics one step further.”

Instead of digging, splashing and scraping to quantify a species’ survival, ecologists can now sample air, water, soil and even the built environment — anywhere a living creature might scrawl its genetic signature with secretions, skin or other scraps of DNA.

From there, researchers isolate any distinct DNA and compare it with known genome sequences. An organism’s DNA can last from a few hours (in the case of certain freshwater crustaceans) to thousands of years (as seen in 13,000-year-old giant ground sloth bone fragments or half-a-million-year-old permafrost-captured horse bones).

This type of genetic sleuthing has helped researchers monitor endangered species, such as Vietnamese crocodile lizards, Australian sea lions, Swedish pool frogs and Canadian lynxes. Last fall, researchers even analyzed eDNA to rule out theories about the mythical Loch Ness monster. (What they found was evidence of lots of eels.)

Because eDNA techniques are less destructive and more efficient than classic surveying methods, they have become popular for examining elusive life-forms: emergent invasives, endangered species or otherwise scarce and secretive creatures.

Take the Rio Grande siren, a cryptic and nocturnal salamander that spends its days hiding in mud. In vain, scientists have baited siren traps with bacon, shrimp and chicken liver, then waited months to catch a single specimen, said Krista Ruppert, a biologist at the University of Texas Rio Grande Valley.

The rediscovery of a fly species in Wales hints at environmental DNA sampling’s potential to change endangered species protection. (Adam McCauley/The New York Times)

“We don’t know much about them because they’re traditionally difficult to study,” Ruppert said. Now, scientists only need to analyze water samples for siren eDNA: “You don’t have to see it to know it’s there.”

The same logic works for tiny invasive species, like nocturnal bloody red shrimp in Lake Erie. Rather than casting thin mesh nets at night and closely examining the contents under a microscope, Penn State researchers last fall detected the species from water samples.

The technology has also proven useful for conservationists grappling with the paradox of extinction: How do you prove a species is truly gone forever?

Unknown unknowns

A scientific expedition to study the depths of the Gulf of Mexico last September highlighted one of the biggest roadblocks to using eDNA methods: incomplete genetic reference databases.

“We end up with a whole lot of sequences,” said Santiago Herrera, a molecular ecologist at Lehigh University who spent a week in September collecting deep-sea eDNA. “But if we don’t recognize them, it’s a lot of questions marks.”

In 2017, European researchers analyzed samples from 18 Finnish stream sites and found that eDNA methods identified more than twice the number of organisms than traditional surveys did. But the team conceded that unreliable reference databases “must be resolved before the full potential of DNA metabarcoding can be unlocked.”

Long-standing scientific biases mean that the genomes of uncharismatic creatures — such as barnacles, scorpions and diatoms — are less likely to be sequenced and identifiable, even if they are vulnerable to extinction. Biologists have described some 1.3 million invertebrates, but that figure only represents a slim fraction of a category of life that includes worms, sponges, insects and mollusks and accounts for about 95% of all animals.

“We are working to populate these databases, but we lose species faster than we have the power to identify them,” said Melania Cristescu, a biologist at McGill University in Montreal.

Scientists are also racing to understand how DNA degrades across different temperatures, microbial communities and levels of acidity and salinity.

“There’s a big leap between what we as scientists can do and how that gets applied in the real world,” said Ivor Knight, a biologist at Penn State who works on detecting bloody red shrimp. “There’s a gap between the understanding of its potential and the understanding of its limitations.”

And there are a number of limitations when it comes to analyzing eDNA. The mere presence of a DNA scrap doesn’t reveal whether it has been there for a day or a millennium, belonged to a horde or an individual or was sloughed off a creature dead or alive.

Even highly trained scientists can accidentally contaminate samples or mistake noisy data as meaningful (and vice versa). When Rogers’ team was confounded by surprising results from a stream near their campus, they realized they probably had detected DNA from the nearby Calgary Zoo.

“Sequencing technology, even though it’s been around for a long time, isn’t perfect,” said Clare I.M. Adams, a biologist at the University of Otago in New Zealand who uses eDNA to investigate the blackfoot pāua sea snail there. “It takes a lot of troubleshooting. And it takes a lot of time and effort to troubleshoot.”

‘Never say die’

Still, ecologists around the world have flocked to the technology — so much so that in 2019 publishing company Wiley launched a peer-reviewed journal dedicated solely to environmental DNA studies.

“It’s growing really, really fast,” said Quentin Mauvisseau, a biologist at the University of Derby in England. “We’ve had a massive increase of people in the field.”

In 2017, after years of using eDNA to study octopuses and crayfish, Mauvisseau turned his attention to the scarce yellow sally stonefly. His search was made possible by Davy-Bowker’s dip in the River Dee earlier that year. About 20 minutes into that day’s expedition, Davy-Bowker captured a living scarce yellow sally stonefly, upending 22 years of presumed local extinction.

“I couldn’t believe it. I was absolutely staggered, really,” he said. “I can’t tell you what a thrill it was to find it again. Never say die.”

He collected additional stoneflies and reared the nymphs to adults, so Mauvisseau could isolate and sequence the DNA sequence of one of the specimens.

“We didn’t have any matches for it,” Mauvisseau said. So he developed an eDNA test that allowed surveyors to return to the River Dee in 2018 with a molecular looking glass.

Using traditional and eDNA sampling across 12 locations on the River Dee, Davy-Bowker and his colleagues documented six sites with traces of the scarce yellow sally stonefly, according to findings published last fall. Davy-Bowker will return in March to survey, and his group plans to eventually, collect, rear and introduce more specimens to more sites in the future.

And if stonefly populations eventually show signs of diminishing once more?